Bottom Line:
In response to a glucose pulse, the cytosolic NADPH/NADP ratio first increased very rapidly and restored the steady state ratio after 3 minutes.In contrast to this dynamic observation, the whole cell NADPH/NADP ratio remained nearly constant.The novel cytosol NADPH/NADP measurements provide new insights into the thermodynamic driving forces for NADP(H)-dependent reactions, like amino acid synthesis, product pathways like fatty acid production or the mevalonate pathway.

Affiliation: Department of Biotechnology, Delft University of Technology, Delft, 2628BC, The Netherlands.

ABSTRACTEukaryotic metabolism is organised in complex networks of enzyme catalysed reactions which are distributed over different organelles. To quantify the compartmentalised reactions, quantitative measurements of relevant physiological variables in different compartments are needed, especially of cofactors. NADP(H) are critical components in cellular redox metabolism. Currently, available metabolite measurement methods allow whole cell measurements. Here a metabolite sensor based on a fast equilibrium reaction is introduced to monitor the cytosolic NADPH/NADP ratio in Saccharomyces cerevisiae: NADP + shikimate â‡„ NADPH + H(+) + dehydroshikimate. The cytosolic NADPH/NADP ratio was determined by measuring the shikimate and dehydroshikimate concentrations (by GC-MS/MS). The cytosolic NADPH/NADP ratio was determined under batch and chemostat (aerobic, glucose-limited, Dâ€‰=â€‰0.1â€‰h(-1)) conditions, to be 22.0â€‰Â±â€‰2.6 and 15.6â€‰Â±â€‰0.6, respectively. These ratios were much higher than the whole cell NADPH/NADP ratio (1.05â€‰Â±â€‰0.08). In response to a glucose pulse, the cytosolic NADPH/NADP ratio first increased very rapidly and restored the steady state ratio after 3 minutes. In contrast to this dynamic observation, the whole cell NADPH/NADP ratio remained nearly constant. The novel cytosol NADPH/NADP measurements provide new insights into the thermodynamic driving forces for NADP(H)-dependent reactions, like amino acid synthesis, product pathways like fatty acid production or the mevalonate pathway.

f7: Model based estimation of the intracellular flux of G6PDH and the split ratio of flux into PPP and glycolysis during the pulse experiments.Data points before tâ€‰=â€‰0 represent the state 5â€‰mins after the SA addition, just before the glucose perturbation.

Mentions:
To obtain an estimation of the dynamic response of the PPP flux, the model of Vaseghi, et al.37 was used. The steady state and dynamic fluxes into PPP and the flux split ratio into glycolysis and PPP are shown in Fig. 7. The detailed calculation is shown in S1 Appendix.

f7: Model based estimation of the intracellular flux of G6PDH and the split ratio of flux into PPP and glycolysis during the pulse experiments.Data points before tâ€‰=â€‰0 represent the state 5â€‰mins after the SA addition, just before the glucose perturbation.

Mentions:
To obtain an estimation of the dynamic response of the PPP flux, the model of Vaseghi, et al.37 was used. The steady state and dynamic fluxes into PPP and the flux split ratio into glycolysis and PPP are shown in Fig. 7. The detailed calculation is shown in S1 Appendix.

Bottom Line:
In response to a glucose pulse, the cytosolic NADPH/NADP ratio first increased very rapidly and restored the steady state ratio after 3 minutes.In contrast to this dynamic observation, the whole cell NADPH/NADP ratio remained nearly constant.The novel cytosol NADPH/NADP measurements provide new insights into the thermodynamic driving forces for NADP(H)-dependent reactions, like amino acid synthesis, product pathways like fatty acid production or the mevalonate pathway.

Affiliation:
Department of Biotechnology, Delft University of Technology, Delft, 2628BC, The Netherlands.

ABSTRACTEukaryotic metabolism is organised in complex networks of enzyme catalysed reactions which are distributed over different organelles. To quantify the compartmentalised reactions, quantitative measurements of relevant physiological variables in different compartments are needed, especially of cofactors. NADP(H) are critical components in cellular redox metabolism. Currently, available metabolite measurement methods allow whole cell measurements. Here a metabolite sensor based on a fast equilibrium reaction is introduced to monitor the cytosolic NADPH/NADP ratio in Saccharomyces cerevisiae: NADP + shikimate â‡„ NADPH + H(+) + dehydroshikimate. The cytosolic NADPH/NADP ratio was determined by measuring the shikimate and dehydroshikimate concentrations (by GC-MS/MS). The cytosolic NADPH/NADP ratio was determined under batch and chemostat (aerobic, glucose-limited, Dâ€‰=â€‰0.1â€‰h(-1)) conditions, to be 22.0â€‰Â±â€‰2.6 and 15.6â€‰Â±â€‰0.6, respectively. These ratios were much higher than the whole cell NADPH/NADP ratio (1.05â€‰Â±â€‰0.08). In response to a glucose pulse, the cytosolic NADPH/NADP ratio first increased very rapidly and restored the steady state ratio after 3 minutes. In contrast to this dynamic observation, the whole cell NADPH/NADP ratio remained nearly constant. The novel cytosol NADPH/NADP measurements provide new insights into the thermodynamic driving forces for NADP(H)-dependent reactions, like amino acid synthesis, product pathways like fatty acid production or the mevalonate pathway.